Viral vectors having chimeric envelope proteins containing the IgG-binding domain of protein A

ABSTRACT

The invention involves viral vectors that can be used to tranduce a target cell, i.e. to introduce genetic material into the cell. The targets of interest are eukaryotic cells and particularly human cells. The transduction can be done in vivo or in vitro. More particularly the invention concerns viral vectors that have chimeric envelope proteins and contain the IgG-binding domain of protein A. These vectors when used in conjunction with antibodies targeting a particular cell are particularly useful for gene therapy.

FIELD OF THE INVENTION

[0001] The invention involves viral vectors that can be used totransduce a target cell, i.e., to introduce genetic material into thecell. The targets of interest are eukaryotic cells and particularlyhuman cells. The transduction can be done in vivo or in vitro. Moreparticularly the invention concerns viral vectors that have chimericenvelope proteins and contain the IgG-binding domain of protein A. Thesevectors when used in conjunction with antibodies targeting a particularcell are particularly useful for gene therapy.

BACKGROUND OF THE INVENTION

[0002] A variety of viral based vectors have been employed to transferand to express a gene of interest into a eukaryotic target cell.Recombinant DNA techniques are used to replace one or more of the genesof the virus with the gene of interest operably linked to a promoterthat is functional in the target cell. The construct, termed a viralvector, infects the target cell, using the physiological infective“machinery” of the virus, and expresses the gene of interest instead ofthe viral genes. Because not all the genes of the virus are present inthe vector, infection of the target by the vector does not produce viralparticles. Viruses that have been used to infect human or mammaliantarget cells include herpes virus, adenovirus, adeno-associated virusand derivatives of leukemia-type retroviruses. Among the retroviruses ofparticular interest in the transduction of cells of human origin areconstructs based on amphotropic retroviruses.

Use of Amphotropic and Ecotropic Retrovirus Vectors

[0003] Retroviruses are particularly well suited for transduction ofeukaryotic cells. The advantages of a vector based this type of virusinclude its integration into the genome of the target cell so that theprogeny of the transduced cell express the gene of interest. Secondly,there are well developed techniques to produce a stock of infectiousvector particles that do not cause the production of viral particles inthe transduced target cell. Lastly, the production and purification ofstocks vector particles having titers of 10⁶ TCIU/ml can beaccomplished.

[0004] One disadvantage of the use of retroviral vectors is that thereis presently no practical general, method whereby a particular tissue orcell type of interest can be specifically transduced. Previous effortsto this end have included surgical procedures to limit to specificorgans the physical distribution of the viral vector particles (Ferry,N. et al., 1991, Proc. Natl. Acad. Sci. 88:8377). Alternatively,practitioners have taken advantage of the fact that type C retrovirusesonly infect dividing cells. Thus, a population of cells, e.g., bonemarrow cells, was removed from a subject and cultured ex vivo in thepresence of growth factors specific for the specific target cell which,thus, comprises most of dividing cells in the culture. See, e.g.,Wilson, J. M. et al., 1990, Proc. Natl. Acad. Sci. 87:439-47; Ohashi, T.et al., 1992, Proc. Natl. Acad. Sci. 89:11332-36. After transduction thedividing cells must be harvested and, for many purposes, reimplantedinto the subject. The technical difficulties of the ex vivo culturetechnique combined with the unavailability of growth factors of specificfor some types of cells have limited the application of this approach.

[0005] A second difficulty presented by the use retroviral based vectorsis that a recombination may occur between sequences of vector and anendogenous retrovirus. Such recombination can give rise to a replicationcompetent virus that can cause the production of infectious particles bythe target cell. In contrast to herpes virus or adenovirus infection,retroviral infections are not necessarily self-limiting.

[0006] Notwithstanding these difficulties, retrovirus vectors based onamphotropic murine leukemia retroviruses that infect human cells, havebeen approved for use in human gene therapy of certain diseases, forexample adenosine deaminase and low density lipoprotein receptordeficiencies and Gaucher's Disease. See, e.g., Miller A. D., 1992,Nature 357:455; Anderson, W. F., 1992, Science 256:808; and Crystal, R.G., 1995, Science 270:404-410.

[0007] One approach to overcoming the limitations of using amphotropicretrovirus vectors in human cells has been to mutate the gene encodingthe protein on the viral surface that determines the specificity ofinfection of the virus, the gp70 protein. Using recombinant DNAtechnology a “mutant” virus is constructed that has had small regions ofthe gp70 sequence replaced by predetermined sequences. The limits ofthis approach are set by the requirement for knowledge of the sequencethat will enable infection of the target of interest. However, when thisknowledge was available, the anticipated alteration in viral specificityhas been observed (Valsesia-Wittmann, S., 1994, J. Virol. 68:4609-19).

[0008] Retrovirus vectors are the most efficient tools available todayto stably transduce genes into the genomes of vertebrate cells. Murineleukemia retrovirus (MLV)-based vectors commonly used for gene transferare classified on the basis of their host range as either ecotropic oramphotropic. Murine ecotropic virions can only infect mouse or ratcells, but murine amphotropic viruses can infect cells of most species,including human cells. Because of their ability to infect such a broadspectrum of cells, a major drawback to the use of amphotropic virusvectors is the fact that these vectors lack target-cell specificity.

[0009] Several attempts to alter the host range of retroviruses havebeen reported to date. Recently, direct modifications of the envelopeprotein of murine leukemia virus (MLV) have been shown to redirect theviral tropism. A recombinant virus containing a fragment encoding asingle Fv antibody chain at the N terminal region of the MLV env genehas been shown to recognize the corresponding epitopes and infect humancells (Russell, S. J. et al., 1993, Nucleic Acids Res. 21:1081-1085;Somia, N. V. et al., 1995, Proc. Natl. Acad. Sci. USA 92:7570-7574;Marin, M. et al., 1996, J. Virol. 70:2957-2962). Kasahara et al. havemade a chimeric ecotropic virus containing an erythropoietin-envelopefusion protein (Kasahara, N. et al., 1994, Science 266:1373-1376). Thischimeric virus has been shown to infect human cells bearing theerythropoietin receptor. However, this type of approach suffers from atleast two limitations. First, each targetable vector must be constructedde novo. It is unlikely that the incorporation of different targetingelements in the envelope of the virus can always be achieved with equalsuccess and without reducing the virus titers than can be obtained.Second, virions constructed to directly bind to specific targets inhuman cells are intrinsically unsafe, as wild-type recombinants couldproduce potentially harmful effects patients treated with such vectors.By contrast, virions constructed as outlined in this manuscript areuninfectious to human cells in the absence of an accompanying targetingreagent, such as a mAb, which is produced separately and only providedin conjunction with the virus at a convenient time.

Known Viral Vector Complexes to Transduce Target Cells

[0010] An alternative to altering the specificity of binding of the gp70protein itself is to employ a second, novel structure that binds or isbonded to both the viral particle and to the target cell. In one exampleof this approach, lactose molecules were covalently coupled, by anon-specific reaction, to the envelope proteins of an ecotropicretrovirus, which does not normally infect human cells. A humanhepatocellular carcinoma that was known to have receptors forlactose-containing proteins was found to be susceptible to transductionby this vector complex, although the integration of the transduced geneof interest in the target cell chromosome was not directly demonstrated(Neda, H. et al., 1991, J. Biol. Chem. 266:14143). No evidence ofexpression was observed in a hepatocellular carcinoma that lacked thelactose specific receptor. The method of Neda results in a variablenumber of binding sites for the exposed acceptor on the target cell,attached to each derivatized or bound envelope protein and, of course,is limited to the case wherein the target cell has a lactose receptor.

[0011] Another approach to targeting is the use of adapter moleculesinvolved an adapter that was not covalently coupled to the vector. Theuse of this type of adapter has been attempted by Roux and hiscolleagues, who have published several reports that relate to thisstrategy (Patent Publication FR 2,649,119 to Piecheczyk, Jan. 4, 1991;Roux P. et al., 1989, Proc. Natl. Acad. Sci. 86:9079-83; Etienne-Julan,M. et al., 1992, J. Gen. Virol. 73:3251-55). Roux and colleagues haveconstructed adapters from two types of proteins, both typicallyantibodies, by biotinylating the proteins and utilizing avidin orstreptavidin tetramer, a protein which binds four biotin molecules, toform aggregates of up to four of the biotinylated proteins.

[0012] A better approach is described in U.S. Ser. No. 08/363,137, filedDec. 23, 1994, Meruelo et al., the contents of which are herebyincorporated by reference into this patent application. Meruelo et al.describe viral complexes and methods of use to prepare pre-formedadaptors and linkers suitable for gen therapy. They are particularlywell-suited for retroviral systems.

Use of Sindbis Virus Vectors

[0013] Sindbis virus, a member of the Alphavirus genus, has receivedconsiderable attention for use as virus-based expression vectors. Manyproperties of alphavirus vectors make them a desirable alternative toother virus-derived vector systems being developed, including rapidengineering of expression constructs, production of high-titered stocksof infectious particles, infection of nondividing cells, and high levelsof expression (Strauss, J. H. et al., 1994, Microbiol. Rev. 58:491-562;Liljeström, P. et al., 1991, Biotechnology 9:1356-1361; Bredenbeek, P.et al., 1992, Semin. Virol. 3:297-310; Xiong, C. et al., 1993, Science243:1188-1191). However, a major drawback to the use of Sindbis virusvectors is the fact that these vectors lack target-cell specificity. Formammalian cells, at least one Sindbis virus receptor is a proteinpreviously identified as the high-affinity laminin receptor, whose widedistribution and highly conserved nature may be in part responsible forthe broad host range of the virus (Strauss, J. H. et al. 1994; Wang, K.S. et al., 1992, J. Virol. 66:4992-5001). It is desirable to alter thetropism of the Sindbis virus vectors to permit gene deliveryspecifically to certain target cell types. This will require both theablation of endogenous viral tropism and the introduction of noveltropism. In the mature Sindbis virus virion, a plus-stranded viralgenome RNA is complexed with capsid protein C to form icosahedralnucleocapsid that is surrounded by lipid bilayer in which two integralmembrane glycoproteins, E1 and E2 are embedded (Strauss, J. H. et al.,1994). Although E1 and E2 form heterodimer that functions as a unit, theE2 domain appears to be particularly important for binding to cells.Monoclonal antibodies (mAbs) capable of neutralizing virus infectivityare usually E2 specific, and mutations in E2, rather than E1, are moreoften associated with altered host range and virulence (Stanley, J. etal., 1985, J. Virol. 56:110-119; Olmsted, R. A. et al., 1986, Virology148:245-254; Polo, J. M. et al., 1988, J. Virol. 62:2124-2133; Lustig,S. et al., 1988 J. Virol. 62:2329-2336). Recently, a Sindbis virusmutant was identified which contained an insertion in E2 and exhibiteddefective binding to mammalian cells. This mutant is expected to beuseful for development of targetable Sindbis virus vectors (Dubuisson,J. et al., 1993, J. Virol. 67:3363-3374).

[0014] Grieve et al. (International Publication No. WO 94/17813published Aug. 18, 1994, “Defective Sindbis Virus Vectors That ExpressToxoplasma Gondii P30 Antigens”) report the use of defective sindbisviral vectors to protect mammals from protozoan parasites, helminthparasites, ectoparasites, fungi, bacteria and viruses, the contents ofwhich are hereby incorporated by reference. Garoff et al. (InternationalPublication No. WO 92/10578 published Jun. 25, 1992, “DNA ExpressionSystems Based On Alphaviruses”) describe the use of alphaviruses toexpress protein sequences for immunization or protein production, thecontents of which are hereby incorporated by reference. Davis et al.(U.S. Pat. No. 5,185,440 issued Feb. 9, 1993, entitled “cDNA CloneCoding For Venezuelan Equine Encephalitis [(VEE)] Virus And AttenuatingMutations Thereof) disclose cDNA encoding VEE and methods of preparingattenuated Togaviruses, the contents of which are hereby incorporated byreference. Huang et al. (U.S. Pat. No. 5,217,879 issued Jun. 8, 1993,entitled “Infectious Sindbis Virus Vectors”) describe infectious Sindbisvirus vectors with heterologous sequences inserted into the structuralregion of the genome, the contents of which are hereby incorporated byreference. Schlessinger et al. (U.S. Pat. No. 5,091,309 issued Feb. 25,1992, entitled “Sindbis Virus Vectors”) describe RNA vectors based onthe Sindbis Defective Interfering (DI) particles with heterologoussequences inserted, the contents of which are hereby incorporated byreference. Dalemans et al. (International Publication No. WO 95/27069published Oct. 12, 1995, “Alpha Virus RNA As Carrier For Vaccines”)report the medical use of alphaviruses, specifically the Semliki ForestVirus, to delivery exogenous RNA encoding a antigenic epitope ordeterminant, the contents of which are hereby incorporated by reference.Dubensky et al., International Publication No. WO 95/07994 publishedMar. 23, 1995, “Recombinant Alphavirus Vectors” describe recombinantretroviral alphavirus vectors for delivery of heterologous genes totarget cells, the contents of which are hereby incorporated byreference. Sjöberg et al., International Publication No. WO 95/31565published Nov. 23, 1995, “Alphavirus Expression Vector” disclose vectorsfor enhanced expression of heterologous sequences downstream from analphavirus base sequence, the contents of which are hereby incorporatedby reference. Liljeström et al., International Publication No. WO95/27044 published Oct. 12, 1995, “Alphavirus cDNA Vectors” describe acDNA construct that may be introduced and transcribed in animal or humancells, the contents of which are hereby incorporated by reference.

SUMMARY OF THE INVENTION

[0015] The invention concerns viral vectors and their use. Specifically,the invention is concerned with viruses having a protein on the viralparticle surface that is a chimeric protein comprising a viral envelopeprotein and an IgG-binding domain of protein A. Because protein A bindsto an Fc region of antibody, these chimeric proteins enable one to usean antibody to target the viral particle to a desired cell to which theantibody binds and not to a cell to which the antibody does not bind.

BRIEF DESCRIPTION OF THE FIGURES

[0016]FIG. 1. A. Schematic representation of expression constructs. p439is the SV40-based expression vector including wild-type Mo-MLV envelopegene. Plasmid p439-ZZ was constructed by replacement of the Mo-MLV envgene with synthetic IgG-binding part (ZZ) of protein A between uniquerestriction sites Bst EII and Bam HI in p439 vector in the presence ofcompatible linker-spacer. See Materials and Methods for details ofconstruction. Abbreviations: LTR, long terminal repeat ; SV40P, SV40early enhancer/promoter; L, leader sequence; SU, surface protein; TM,transmembrane protein; ZZ, synthetic protein A; L/S, Linker-Spacer;p(A), polyadenylation signal. B. Immunoblot analysis of lysates fromCOS-7 cells transiently transfected with p439 and p439-ZZ. Lane 1 and 2were stained with a SU antiserum followed by HRP-conjugated rabbitanti-goat IgG. Lane 3 and 4 were stained with HRP-conjugated rabbit IgGfor detection of protein A.

[0017]FIG. 2. A. Immunoblot analysis of virions produced by ψ2 andψ2-ZZ10 packaging cells. Lane 1 and 2 were stained with a SU antiserumfollowed by HRP-conjugated rabbit anti-goat IgG. Lane 3 and 4 werestained with HRP-conjugated rabbit IgG for detection of protein A. B.ELISA for detection of IgG-binding activity of chimeric virus producedby ψ2-ZZ10 cells. Open circle, virions from ψ2; closed circle, virionsfrom ψ2-ZZ10. Results are average of triplicate determinants.

[0018]FIG. 3. (A) Schematic strategy for retargeting an Sindbis virusvector. A wild-type Sindbis virus (left) binds to mammalian cells viaits surface receptor which is known to be highly conserved acrossspecies. A recombinant Sindbis virus displaying IgG-binding domain ofprotein A (right) should permit binding to a novel target molecule onthe cell surface when used with a corresponding monoclonal antibody(mAb). (B) Schematic representation of recombinant helper constructs anda SinRep/LacZ expression vector. DH-BB is a parental helper plasmidwhich contains the genes for the structural proteins (capsid, E3, E2, 6Kand E1) required for packaging of the Sindbis viral genome. DH-BB-Bstwas constructed by introduction of a cloning site (BstEII) into the E2glycoprotein between amino acids 71 and 74. The synthetic IgG-bindingdomain (ZZ) of protein A was inserted at BstEII in the DH-BB-Bst helperplasmid and DH-BB-ZZ was obtained. SinRep/LacZ, is a Sindbis virus-basedexpression vector which contains the packaging signal, nonstructuralprotein genes for replicating the RNA transcript and lacZ gene.Abbreviations: P_(SG), Sindbis viral subgenomic promoter; C, capsid;nsP1-4, nonstructural protein genes 1-4; ZZ, synthetic IgG-bindingdomain of protein A; p(A), polyadenylation signal.

[0019]FIG. 4. Detection of Sindbis viral structural protein componentsand a recombinant envelope. Cell lysates (A) from BHK cells transfectedwith helper RNA and pellets of viral particles (B and C) produced fromthese cells were subjected to SDS-PAGE analysis. After transferring to anitrocellulose filter, viral proteins were stained with dilutedanti-Sindbis virus mouse immune ascitic fluid to detect all structuralcomponents (A and B) or with HRP-conjugated goat anti-mouse IgG todetect protein A-envelope chimeric protein (C). In each panel, lane 1,DH-BB; lane 2, DH-BB-Bst; lane 3, DH-BB-ZZ.

[0020]FIG. 5. Infection of HeLa and HeLa-CD4⁺ cells with recombinantSindbis virus derived from DH-BB-ZZ helper RNA which is transducing thebacterial lacZ gene. Viral supernatants (200 μl) were preincubatedwithout or with anti-CD4 mAb (0.5 μg/ml) at room temperature for 1 hour,and added to each cells (2×10⁵) in 6-well plates. After 1 hourincubation at room temperature, cells were washed with PBS and incubatedin growth medium for 24 hours. Viral infection was evaluated by X-GalStaining.

[0021]FIG. 6. Antibody-dependent infectivities of recombinant Sindbisvirus particles on A431 and U87MG cells. Viral supernatants (20 μl forDH-BB, 500 μl for DH-BB-ZZ) were preincubated without or with anti-EGFRmAb (0.5 μg/ml) at room temperature for 1 hour, and added to cells(2×10⁵) in 6-well plates. After 1 hour incubation at room temperature,cells were washed with PBS and incubated in growth medium for 24 hours.Viral infection was evaluated by X-Gal Staining.

[0022]FIG. 7. Antibody-dependent infectivities of recombinant Sindbisvirus particles on suspension cells Daudi and HL-60. Viral supernatants(500 μl) derived from DH-BB and DH-BB-ZZ transfected BHK cells werepreincubated without or with 0.5 μg/ml of mAbs (anti-HLA-DR for Daudiand anti-CD33 for HL-60) at room temperature for 1 hour, and added tocells (1×10⁶) in 6-well plates. After 1 hour incubation at roomtemperature, cells were washed with PBS and incubated in growth mediumfor 24 hours. Control shows uninfected cells. Viral infection wasevaluated by FACS-Gal analysis described in Experimental protocol.Positive percent of infected cells were shown in each panel.

DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention provides for a means for modifying theexpression of genes in eukaryotic cells, such as mammalian cells oravian cells, and, more particularly, of human cells for medical practiceand also of the cells of domesticated animals that are valuable foragriculture and recreational purposes for veterinary practice. Theinvention provides for the introduction and expression of geneticmaterial into the cells by means of a viral vector complex. In the viralvector, some or all of the viral genes have been replaced by a gene thatis to be expressed in the eukaryotic target cell. The essential viralgenes that have been removed from the vector are, in general, insertedinto the genome of the cell line that is used to produce stocks of theviral particles. The producer cells lines thus complement the defectsthat are present in the viral vector. In some embodiments, the onlyviral gene contained in the genome of the vector is a gene that isneeded for the packaging of the vector genome into the viral particles.

[0024] Specifically, the invention is directed to viral vectors fortransducing a target cell encoding a chimeric protein comprising anenvelope protein and an IgG-binding domain of protein A. In oneembodiment the envelope protein is a retroviral envelope protein. Anexample of may be Moloney MLV envelope protein. In the envelope proteinis inserted the IgG binding domain of protein A. As used herein, proteinA may be a portion of native protein A or synthetic protein having theFc binding ability of native protein A. In one embodiment it is insertedinto the hypervariable region of gp70.

[0025] In an alternative embodiment the envelope protein is analphavirus envelope protein. An example of an alphavirus may be aSindbis virus. For the Sindbis virus it is preferable to insert theprotein A into the E2 domain. The protein A is preferably inserted so asto reduce or minimize the non-specific infectivity of the Sindbis virus.One example of an insertion site is the position between amino acids 71and 74 of the E2 glycoprotein.

[0026] The construction of viral-based vectors suitable for the generalexpression of genes in cells that are susceptible to infection by thevirus is described the following patent publications: WO 89/05345 toMulligan, R. C. and others, WO 92/07943 to Guild, B. C. and othersconcerning retroviral vectors; WO 90/09441 and WO 92/07945 to Geller, A.I. and others concerning herpes vectors; WO 94/08026 to Kahn, A. andothers, and WO 94/10322 to Herz, J. and others concerning adeno virusvectors; U.S. Pat. No. 5,354,678 to Lebkowski and U.S. Pat. No.5,139,941 to Muzcyzka concerning adeno-associated virus; and U.S. Pat.No. 5,217,879 to Huang et al. and U.S. Pat. No. 5,091,309 to Schlesingerconcerning Sindbis viral vectors. Packaging systems for the productionof retroviral vectors have been described by Danos, O. et al., 1988,Proc. Natl. Acad. Sci. 85:6460-64, and by Landau, N. R. et al., 1992, J.Virol. 66:5110-13, the contents of the above are hereby incorporated byreference.

[0027] The complexes described herein can be provided with a variety ofspecificities. The application discloses methods of constructing acomplex comprising an antibody specific for an acceptor on the targetcell so that the vector complex are internalized into the target cellafter the vector complex is bound. There are a large number of cellsurface antigens suitable for use as acceptors and for which antibodiesare already available. Such structures include, but are not limited to,the class I and class II Major Histocompatibility Antigens; receptorsfor a variety of cytokines and cell-type specific growth hormones, brainderived neurotrophic factor (BDNF), ciliary neurotrophic factor (CTNF),colony stimulating growth factors, endothelial growth factors, epidermalgrowth factors, fibroblast growth factors, glially derived neurotrophicfactor, glial growth factors, gro-beta/mip 2, hepatocyte growth factor,insulin-like growth factor, interferons (α-IFN, β-IFN, γ-IFN, consensusIFN), interleukins (IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,IL-9, IL-10, IL-11, IL-12, IL-13, IL-14), keratinocyte growth factor,leukemia inhibitory factors, macrophage/monocyte chemotactic activatingfactor, nerve growth factor, neutrophil activating protein 2, plateletderived growth factor, stem cell factor, transforming growth factor,tumor necrosis factors and vascular endothial growth factor; celladhesion molecules; transport molecules for metabolites such as aminoacids; the antigen receptors of B- and T-lymphocytes; and receptors forlipoproteins. The invention makes possible the specific infection of acell type by allowing the employ of differentiation antigens as targetsfor the viral vector complex.

[0028] The invention is used to transduce a gene of interest into atarget cell. In practicing the preferred embodiment of the invention,the viral vector and the antibody are preincubated prior to contactingthe target cell acceptor.

[0029] The practice of the invention can be performed by culturing thetarget cells ex vivo. The cultured cells can be continued in culture toproduce the product encoded by the transduced gene. Alternatively, theex vivo transduced cell can be implanted into a subject, which can bethe host from which the cultured cells were obtained.

[0030] In a yet further embodiment, the viral vector and appropriateantibodies can be administered directly to the subject thereby obviatingthe need for any ex vivo cell culture. The routes of administration tothe subject can be any route that results in contact between the vectorcomplex and the target cell. Thus for example, intravenousadministration is suitable for target cells in the hepatic, splenic,renal cardiac and circulatory or hematopoietic systems. The vectorcomplex can also be administered by catheterization of the artery orvein leading to the target organ, thereby allowing the localizedadministration of the complex. The complex can also be administered byinspiration when the target cells are in the respiratory system.

[0031] Genes that can be transduced by the practice of the inventioninclude any gene that can be expressed in a eukaryotic system.Illustrative examples of genes that can be expressed by use of thepresent invention include glucocerebrosidase, adenosine deaminase, andblood coagulation factors such as factor VIII and factor IX.

[0032] The viral component of the vector complex can be based on anyvirus, the particles of which are unable to bind or have been modifiedto be unable to bind to cells of the same species as the target cell. Anon-limiting example of the virus are the murine ecotropic leukemiaretrovirus viruses, e.g., Moloney Leukemia Virus or AKV. Alternatively,chemically modified viral particles can be employed. In addition toecotropic retroviruses, viruses that can be employed to constructvectors according to this embodiment of the invention includeamphotropic retrovirus, herpes virus, adenovirus and adeno-associatedvirus. In addition, the viral component may be an alphavirus, such as aSindbis Virus.

[0033] The viral vectors and viral complexes of the invention may beused to treat a variety of disorders in man and animals. The vectorsbased on the Sindbis virus are particularly well suited forintracellular vaccination. That is, the viral complex carries with it agene of interest encoding a particular antigen. The viral complex willbe taken up into the cell and the gene of interest encoding the antigenis will be expressed in the cellular cytoplasm. By targeting the viralcomplex to desired cellular target, the antigen will be expressed withinthe cell of interest.

[0034] The complexes of this invention are also well suited for thedelivery of antisense sequences.

[0035] There are many examples of bacterial and viral diseases that maybe prevented or ameliorated by the methods described herein.Specifically, the methods described herein may be used for the followingdiseases: adenovirus, AIDS, antibiotic associated diarrhea, bacterialpneumonia, bovine herpes virus (BHV-1), chlamydia, croup, diphtheria,Clostridium difficile, cystitis, cytomegovirus (CMV), gastritis,gonorrhea, heliobactor pyliori, hepatitis A, hepatitis B, herpes virus,HSV-1, HSV-2, human papilloma virus, influenza, legionnaires disease,Lyme disease, malaria, multiple sclerosis, peptic ulcer, pertussis,psoriasis, rabies, respiratory syncytial virus (RSV), rheumatoidarthritis, rhinovirus, rotovirus, salmonella, Stomach cancer, strepthroat, tetanus or travelers diarrhea.

[0036] The embodiments of the invention are described in greater detailhereinafter.

EXAMPLES Example 1

[0037] In this example we describe the construction of a recombinantecotropic retrovirus displaying protein A-envelope chimeric proteins.Protein A, a protein derived from Staphylococcus aureus, has a strongaffinity for the Fc region of various mammalian IgGs (Surolia, A. etal., 1982, Trends Biochem. Sci. 7:74-76). Native protein A has fivehomologous IgG-binding domains (E, D, A, B and C) , and we have utilizedthe synthetic Z domain which is based on the B domain of protein A(Nilsson, B. et al., 1987, Protein Eng. 1:107-113). The development ofretroviral vectors that can bind IgGs (monoclonal antibodies) would haveimportant applications for specific gene delivery. Materials and methods

Plasmids and Cell Line

[0038] A SV40-based plasmid, p439 (SV-E-MLV-env), which express MoloneyMLV (Mo-MLV) envelope protein (Landau, N. R. et al., 1992, J. Virol.66:5110-5113), was kindly provided Dr. Dan R. Littman, New YorkUniversity. pEZZ 18, which contains two synthetic Z domains based on theB domain of protein A (Löwenadler, B. et al., 1987, Gene 58:87-97) waspurchased from Pharmacia Biotech, Uppsala, Sweden. pZeoSV, which hasZeocin-resistant gene for selection, was purchased from Invitrogen Co.,San Diego, Calif. An ecotropic retroviral packaging cell line ψ2 (ATCCCRL9560) (Mann, R. et al., 1983, Cell 33:153-159) and COS-7 cells (ATCCCRL1651) were maintained in Dulbecco's modified Eagle's medium (DMEM)supplemented with 10% fetal bovine serum (FBS).

[0039] 6.1.2. Construction of chimeric env gene

[0040] Two synthetic IgG-binding domain of protein A (ZZ) were amplifiedby polymerase chain reaction (PCR) using PEZZ 18 as a template. Primersused for PCR amplification are ZZ-5(5′-CACGATGAGGTAACCGACAACAAATTCAAC-3′) (SEQ ID NO. 1), with Bst EIIsite, and M13 (−40) sequencing primer (5′-GTTTTCCCAGTCACGAC-3′) (SEQ IDNO. 2) which locates downstream from the multiple cloning sites of pEZZvector. The resulting PCR products were digested with Bst EII and Eco RIand replaced the Mo-MLV env gene between unique restriction sites BstEII (position 5923) and Bam HI (position 6537) of the p439 vector in thepresence of compatible oligonucleotides EB1(5′-AATTCGGGAGGCGGTGGATCAGGTGGAGGCGGTTCAGG-3′) (SEQ ID NO. 3) and EB2(5′-GATCCCTGAACCGCCTCCACCTGATCCACCGCCTCC-3′) (SEQ ID NO. 4) to act as alinker-spacer. Clones containing inserts of proper size were sequencedto confirm that the correct reading frames were maintained.

Cell transfection and virus production

[0041] The wild-type and protein A-gp70 chimeric envelope genes werefirst transiently transfected into COS-7 cells. 2×10⁵ cells were seededin 3.5 cm-diameter dishes and transfected the next day with 2 μg ofplasmid with 10 μl of LipofectAmine reagent (Gibco-BRL, Gaithersburg,Md.). 72 h after transfection, cells were collected and subjected toimmunoblot analysis. To create packaging cell lines expressing therecombinant envelope, 5×10⁵ ψ2 cells were transfected with 20 μg ofchimeric envelope plasmids and 1 μg of pZeoSV by the CaPO₄ method(Stratagene, La Jolla, Calif.) (Mann, R. et al., 1983). The medium waschanged 16 hours later and transfected cells were selected with 250μg/ml of Zeocin (Invitrogen Co., San Diego, Calif.) After selection for10 days, Zeocin-resistant cell colonies were picked for expansion andscreened by immunoblot analysis and ELISA as described below.

Immunoblot assay

[0042] For monitoring of protein A-envelope chimeric protein expression,transfected cells and viral samples were subjected to immunoblotanalysis. Virus samples were pelleted by ultracentrifugation of thesupernatants (10 ml) in an SW41 Beckmann Rotor (25,000 rpm, 2 h, 4° C.).Immunoblot analysis was performed as described before (Marin, M. et al.,1996, J. Virol. 70:2957-2962) by using a goat antiserum against Rausherleukemia virus SU protein (Quality Biotech Inc., Camden, N.J.) andhorseradish peroxidase-conjugated rabbit anti-goat IgG antibodies(Pierce, Rockford, Ill.).

ELISA

[0043] ELISA was performed to detect chimeric virus carrying proteinA-envelope chimeric protein in the culture supernatants. Briefly,pelleted viral particles from 10 ml culture supernatants wereresuspended in 400 μl of phosphate buffered saline. 96-well microtiterplates (Dynatech Laboratories, INC., Chantilly, Va.) were coated withduplicate serial dilutions of viral samples for 2 h at RT followed byblocking with PBS containing 1% BSA and 0.05% Tween 20. Then 0.1 μg/mlof horseradish peroxidase-conjugated rabbit anti-goat IgG antibodies(Pierce) was added to each well and incubated for 2 h at RT. Afterwashing with PBS containing 0.05% Tween 20, the binding activity of eachwell was determined by using o-Phenylenediamine (Pierce) as a substrate.

Results

[0044] Plasmid construction and transient expression in COS-7 cells

[0045] A modified Mo-MLV envelope expression vector, p439-ZZ, that wouldexpress two synthetic IgG-binding domain of protein A was generated(FIG. 1). The position of replacement in gp70 was previously shown toallow the functional display of erythropoietin (Kasahara, N. et al.,1994) and heregulin (Han, X. et al., 1995, Proc. Natl. Acad. Sci. USA92:9747-9751). The C-terminus of the protein A gene is connected to aproline rich hypervariable region of gp70 with the EB linker-spacer(SGGGGSGGGGS) (SEQ ID NO. 5) in order to avoid interactions between theIgG-binding part of protein A and the envelope protein segment of therecombinant fusion protein. Expression is driven by the SV40 earlyenhancer/promoter sequence and the 5′ long terminal repeat (LTR). Theplasmid p-439-ZZ was deposited with the American Type Culture Collection(ATCC) on Mar. 28, 1997.

[0046] To examine the expression of the recombinant envelope, wetransfected p439-ZZ expression plasmid into COS-7 cells. Lysates fromtransfected and nontransfected cells were first analyzed for envelopeexpression by using anti-Rauscher leukemia virus SU serum whichcross-reacts ecotropic (70 kDa) Mo-MLV SU protein. As expected, thewild-type p439 plasmid expressed major protein bands of gp70 and itsprecursor (80 kDa) (FIG. 1B, lane 2). The recombinant p439-ZZ plasmidexpressed immunoreactive proteins at 70 kDa corresponding to precursorprotein of the recombinant envelope suggesting that protein A-gp70 couldbe expressed in transfected COS cells. The same lysates were used fordetection of IgG-binding activity using Horseradishperoxidase-conjugated rabbit anti-goat IgG. As shown in FIG. 1B, lane 6,the protein A-gp70 chimeric envelope precursor at 70 kDa expressed byp439-ZZ plasmid showed IgG-binding activity. Stable expression of thechimeric protein A-gp70 protein suggests that the protein A domain wasproperly folded after translation.

[0047] Creation of packaging cell lines producing protein A-envelopechimeric virus.

[0048] The chimeric envelope plasmid, p439-ZZ, and Zeocin-resistancegene were cotransfected into ψ2 packaging cell line, which expressesgag, pol and env gene products of E-MLV. After selection with Zeocin,subclones were isolated and screened for protein A-gp70 expression byimmunoblot analysis of whole cell lysate using rabbit IgG. One subclone,designated ψ2-ZZ10, showed cytoplasmic IgG-binding activity and waschosen for further characterization. To demonstrate the incorporation ofthe chimeric envelope protein into virions, retroviral particles werepurified by sucrose density gradient centrifugation. The viral pelletswere then subjected to immunoblot analysis with anti-Rauscher leukemiavirus SU serum or rabbit anti-goat IgG. Major bands of 70 kDa, whichwere derived from wild-type env gene of ψ2 packaging cells, could bedetected in both virions from ψ2 and ψ2-ZZ10 cells (FIG. 2A, lane 1 and2). The band of 60 kDa, which was estimated MW of protein A-gp70chimeric protein, was also detected in virions produced by ψ2-ZZ10.However, less chimeric envelope was found in virus pellet compared withwild-type envelope. Virions produced by ψ2-ZZ10 showed IgG-bindingactivity at the band of 60 kDa whereas there was no IgG-binding activityin that of untransfected ψ2 cells (FIG. 2A, lane 3 and 4). TheIgG-binding activity of chimeric virus was further confirmed by ELISA.As shown in FIG. 2B, the protein A-envelope chimeric virus produced byψ2-ZZ10 cells exhibited IgG-binding activity in a concentrationdependent manner compared with that of untransfected ψ2 cells. Takentogether, these results demonstrate that p439-ZZ produces recombinantretrovirus displaying the IgG-binding domain in its envelope.

Discussion

[0049] In this study we have shown that protein A can be displayed onthe surface of murine ecotropic retroviral particles fused to the nativeenvelope protein. The protein A-gp70 chimeric protein derived fromp439-ZZ was correctly expressed and incorporated into virions.Furthermore, IgG-binding activity was detected in virions produced byψ2-ZZ10 cells. In this study the chimeric envelope did not express asefficiently as that of wild type envelope in virions produced by ψ2-ZZ10(FIG. 2A). We are currently trying to increase the expression of proteinA-gp70 protein by changing the enhancer/promoter of the expressionplasmid as well as utilizing other packaging cell lines.

[0050] The use of antibody-antigen interactions as the basis fortargeting has a great advantage because a number of monoclonalantibodies have been developed and investigated. Since the protein Aportion of the chimeric envelope binds to the Fc domain of the antibody(Surolia, A. et al., 1982), it allows flexibility with regard to thetargeting elements, as any of a variety of mAbs can be selected. It hasbeen reported that the binding of retrovirus-associated antibodyfragments to the cell surface is followed by membrane fusion betweenvirus and target cells (Etienne-Julan, M. et al., 1992, Roux, P. et al.,1989). The protein A-envelope chimeric retrovirus displaying mAbsagainst cell surface antigens should bind preferentially to target cellsexpressing those antigens, and this may facilitate their infection.

[0051] Furthermore, in principle, a similar approach may be used withother viral vectors, such as adenovirus and Sindbis virus vectors byinserting the synthetic IgG binding domain (ZZ) of protein A. We alsohave constructed a recombinant Sindbis virus vector with proteinA-envelope and demonstrated its high efficient cell-specific infectionagainst variety of human cells, see Example 2. The protein A-enveloperetroviral vector as described in this example should also permitinfection against specific cell types once the expression of chimericenvelope successfully increased in the virion. In conclusion, the novelcell targeting system which utilizes protein A-mAb interaction for virusinfection would have broad applications for gene expression studies andtherapy.

Example 2

[0052] In this example we describe the construction of a recombinantSindbis virus vector displaying protein A-envelope chimeric proteins toredirect the viral tropism. Protein A (PA), a protein derived fromStaphylococcus aureus, has a strong affinity for the Fc region ofvarious mammalian IgGs (Surolia, A. et al., 1982). In contrast to thetargeted retroviral vectors described above, the PA-envelope chimericvirus vector once successfully generated needs no further modificationto target distinct cells. The targeting is achieved simply by changingthe complementary mAb (FIG. 3A). More importantly, we demonstrate thatthis chimeric virus used in conjunction with mAbs can infect human cellsand transfer a test gene, bacterial β-galactosidase with highefficiency. The novel cell targeting system which utilizes PA-mAbinteraction for virus infection would have important applications forgene expression studies and therapy.

Results Construction of protein A-envelope Sindbis virus helper plasmid

[0053] To modify the Sindbis virus envelope protein, we have utilizedthe DH-BB helper plasmid (FIG. 3B) which was constructed by deletion ofthe region between BspMII and BamHI sites of the full-length Sindbisvirus cDNA clone (Bredenbeek, P. J. et al., 1993, J. Virol.67:6439-6446). When RNA from DH-BB is cotransfected with recombinant RNAfrom the Sindbis virus expression vector (for example, SinRep/LacZ, FIG.3B), the structural proteins expressed in trans, from the DH-BB RNAtranscript allows packaging of the recombinant RNA into virions. SinceDH-BB does not contain a packaging signal, it will not form a defectiveinterfering particle or be packaged with recombinant RNA. Two modifiedSindbis virus helper plasmids were constructed: DH-BB-Bst into which aBstEII cloning site was inserted and DH-BB-ZZ into which two IgG-bindingdomain of PA were inserted in the E2 region, were generated (FIG. 3B).Native protein A has five homologous IgG-binding domains (E, D, A, B andC) , and we have utilized the synthetic Z domain which is based on the Bdomain of protein A (Nilsson, B. et al., 1987). The insertion position,between codons 71 and 74 amino acids in E2, was chosen because mutationsin this region were previously shown to allow normal particle assemblyand release block virus entry at the level of attachment (Dubuisson, J.et al., 1993).

Expression and incorporation of chimeric envelopes into virions

[0054] After linearization of helper and SinRep/LacZ plasmids, in vitrotranscription was performed and the quality of RNA was checked onagarose gels (data not shown). To examine the expression of therecombinant envelope, recombinant helper RNA was cotransfected with RNAfrom SinRep/LacZ plasmid into BHK cells by electroporation. Thetransfection efficiency was usually nearly 100% under the proceduredescribed in Experimental protocol below (data not shown). Lysates fromtransfected cells were first analyzed for expression of structuralprotein by using anti-Sindbis virus immune ascitic fluid. As shown inFIG. 4A, DH-BB-Bst helper RNA expressed a 50-55 kDa band of envelope (E1and E2) and a 33 kDa of capsid protein which is the same protein profileas the parental virus produced by DH-BB. A band of 60 kDa correspondingto the E2 precursor PE2 was also detected. In the protein profileexpressed by DH-BB-ZZ RNA, a major band between 65-70 kDa, which is theestimated MW of PA-E2 and PA-PE2 chimeric protein, was observed as wellas the 33 kDa capsid protein. These results suggest that the mutantswere correctly expressed and processed. A band of envelope (E1) looksslightly shifted below in the lysate from DH-BB-ZZ transfected cells dueto the disappearance of E2 glycoprotein.

[0055] To demonstrate the incorporation of the chimeric envelope proteininto virions, viral pellets were subjected to immunoblot analysis. Asshown in FIG. 4B, virions produced by DH-BB and DH-BB-Bst RNA containcapsid and envelope (E1 and E2) proteins indicating that the mutation inDH-BB-Bst does not affect virus assembly. The PA-E2 chimeric protein wasalso incorporated into virions and exhibited IgG-binding activity whichis not detected in that of DH-BB and DH-BB-Bst (FIG. 4B and C). Theseresults demonstrate that DH-BB-ZZ produces recombinant Sindbispseudovirions displaying the IgG-binding domain in its envelope. Theprotein band of E1, which was expressed in transfected cells (FIG. 4A,lane 3) could not be detected in the virions produced by DH-BB-ZZ RNA.

Infection with viruses carrying mutant envelopes

[0056] Infectivities of recombinant viruses against hamster and humancells were determined by transfer of the Sindbis virus vector(SinRep/LacZ) that can transduce bacterial β-galactosidase gene. Asshown in Table 1, viruses derived from DH-BB and DH-BB-Bst helper showedvery high infectious titer (10⁸ LacZ CFU/ml) against BHK cells whereasviruses produced by DH-BB-ZZ showed very low infectivity (10³ LacZCFU/ml) suggesting that the protein A insertion into E2 blocked virusbinding to host cells supporting previous observations (Dubuisson, J. etal., 1993). The PA-envelope virus also showed minimal titer againsthuman HeLa-CD4⁺ cells (10² LacZ CFU/ml). When virions were preincubatedwith anti-CD4 mAb, however, the protein A-envelope chimeric virus couldinfect HeLa-CD4⁺ cells in a antibody dose-dependent manner (Table 1).When the viral supernatant was preincubated with 0.5 μg/ml mAb, aninfectious titer was approximately 10⁵ LacZ CFU/ml. The enhancement ofinfectivities by mAb was not observed with that of DH-BB and DH-BB-Bstderived viruses. As shown in FIG. 5, the protein A-envelope chimericvirus with anti-CD4 mAb could not infect HeLa cells which do not expressCD4 on its surface indicating that the infection is dependent on both anantibody and a corresponding antigen. These data demonstrate that thePA-E2 chimeric envelope derived from DH-BB-ZZ helper RNA can redirectSindbis virus infection via a new receptor/antigen in the presence ofrecognizing antibody.

[0057] Next, we determined whether PA-E2 displaying virus particles werecapable of infection against various human cell lines expressingspecific antigens on their surface. For adherent cells, epidermoidcarcinoma cell line A431 and glioblastoma cell line U87MG, bothoverexpressing epidermal growth factor receptors (EGFR), were used. Asexpected, viruses with PA-envelope could infect these cells efficientlyonly when virions were preincubated with anti-EGFR mAb (FIG. 6).Infectious titers of the recombinant virus with mAb (0.5 μg/ml) againstA431 and U87MG cells were approximately 10⁴ LacZ CFU/ml. Again, minimalinfectivities (10² LacZ CFU/ml) were seen on these cells when infectedwithout mAb. We next used two human suspension cell lines, Burkitt'slymphoma cells, Daudi, and promyelocytic leukemia cells, HL-60. In thisexperiment infected cells were detected by FACS-Gal analysis. TypicalFACS results of infectivity are presented in FIG. 7. In contrast to thedata with adherent cells (FIG. 6), the wild-type virus particles derivedfrom DH-BB helper RNA have very low infectivities against Daudi andHL-60 cells. However, the PA-envelope virus preincubated withcorresponding mAbs (anti-HLA-DR for Daudi and anti-CD33 for HL-60) couldinfect these cells with very high efficiency, and the positive percentof infected cells were more than 90% in both cell lines. Infection bythe protein A-envelope virus of these cells was not observed in theabsence of mAb.

Discussion

[0058] In this invention we describe the construction of a recombinantSindbis virus vector displaying protein A-envelope chimeric proteins onthe viral surface. The synthetic IgG-binding domain of protein A (ZZ) atthe position between 71 and 74 amino acids of the E2 glycoprotein; thissite has been shown to block Sindbis virus binding to host cells(Dubuisson, J. et al., 1993). The PA-E2 chimeric protein was correctlyexpressed and incorporated into Sindbis virions and exhibitedIgG-binding activity as shown in FIG. 4B and C. In this experiment,however, the incorporation of E1 glycoprotein into virions could not bedetected (FIG. 4C, lane 3) although it is expressed in transfected cells(FIG. 4A, lane 3). Insertion of the IgG-binding domain producesstructural change of recombinant E2 chimeric protein that inhibits itsinteraction with E1 to form a heterodimer. The interaction between E1and PA-E2 protein is not fully understood. This result also indicatesthat Sindbis virus assembly may occur without incorporation of the E1glycoprotein. This observation may provide insight into mechanism ofSindbis virus assembly.

[0059] The PA-envelope chimeric Sindbis virus vector showed minimalinfectivities against BHK and other human cell lines. However, when usedin conjunction with mAbs which react with cell surface antigens, thePA-envelope chimeric virus was able to transfer the LacZ gene into humancell lines with high efficiency. The new tropism of the recombinantvirus depends on antigen-antibody interaction since the PA-envelopevirus could not infect targeted cells without mAb and correspondingantigen on cell surface (FIG. 5). Taken together, the PA-E2 chimericenvelope derived from DH-BB-ZZ helper RNA can redirect Sindbis virusinfection with high efficiency by antigen-antibody interaction.

[0060] Several retrovirus and adenovirus-based cell-targeting vectorshave been developed recently (Russell, S. J. et al., 1993; Somia, N. V.et al., 1995; Marin, M. et al., 1996; Douglas, J. T. et al., 1996,Nature, Biotechnology 14:1574-1578). The novel cell-targeting systemdeveloped in this study has some advantages compared with theseretroviral and adenoviral retargeting vectors. In this approach it isnot necessary to construct each targetable vector de novo. It isunlikely that the incorporation of different targeting elements in theenvelope of the virus can always be achieved with equal success andwithout reducing the virus titers that could be obtained. Since theprotein A portion of the chimeric envelope binds to the Fc domain of theantibody (Surolia, A. et al., 1982), it allows flexibility with regardsto the targeting elements, as any of a variety of mAbs can be selected.In addition, replication occurs entirely in the cytoplasm of theinfected cells as an RNA molecule, without a DNA intermediate (Strauss,J. H. et al., 1994). This is in contrast to retrovirus vectors, whichmust enter the nucleus and integrate into the host genome for initiationof vector activity. Thus, retrovirus-derived vectors have applicationsfor long-term expression of foreign proteins, while alphavirus vectorsare useful primarily for transient high-level expression. Furthermore,although adenovirus vectors can express high levels of foreign proteins,these systems are far more complex than alphaviruses and express manyhighly antigenic virus-specific gene products including structuralproteins (Rosenfeld, M. A. et al., 1991, Science 252:431-434). Incontrast, current alphavirus vectors express only the four viralreplicase proteins (nonstructural proteins nsP1 through nsP4) requiredfor RNA amplification in the transduced cells.

[0061] There are several issue which have to be considered in workingwith Sindbis vectors. First, Sindbis virus infection of vertebrate cellsusually results in cell death by apoptosis (Levine, B. et al. 1993,Nature 361:739-742), with the notable exception of neuronal cells inwhich a persistent infection may be established (Levine, B. et al. 1992,J. Virol. 66:6429-6435). Although this cytotoxicity may be suitable forgene therapy for cancer, long-term or inducible expression vectors wouldhave broader application. It has been reported that the transformationof cells with the cellular oncogene bcl-2 led to a cell line in whichSindbis virus no longer induces apoptosis and instead establishes apersistent infection (Levine, B. et al., 1993; Levine, B. et al., 1996,Proc. Natl. Acad. Sci. USA 93:4810-4815, the contents of which arehereby incorporated by reference into the present application). bcl-2may be used to construct a long-term Sindbis virus expression vectorthat overcomes the problems of apoptosis. The bcl-2 vector would beparticularly well suited to create a master packaging cell line alsoexpressing the both chimeric Sindbis envelop protein and a heterologousgene of interest under the control a Sindbis promotor. Second, therecombinant Sindbis virus vector developed in this invention may havelow infectivities even in the absence of antibody. Accordingly, theremight be other sites in E2 or E1 which are involved in receptor binding(Strauss, J. H. et al., 1994). Furthermore, different receptors havebeen identified on chicken embryo fibroblast (Wang, K. S. et al.,Virology 181:694-702) and mouse neuronal cells (Ubol, S. et al., 1991,J. Virol. 65:6913-6921), suggesting that the Sindbis virus can utilizemore than one receptor. For safety reason, it is desirable to developimproved recombinant Sindbis virus vector which do not infect anymammalian cells when not used with mAbs.

[0062] This invention represents the first demonstration of theretargeting of a Sindbis virus vector by a novel utilization of theprotein A-antibody interaction. A similar approach may be used withother viral vectors, such as retrovirus and adenovirus vectors byinserting the synthetic IgG binding domain (ZZ) of protein A. Thevirus-based vectors displaying protein A-envelope could be very usefuland have a broad applicability for gene transfer study and for the genetherapy field.

Experimental protocol

[0063] Cell lines. Baby hamster kidney (BHK) cells were obtained fromInvitrogen Co., San Diego, Calif., and maintained in minimum essentialmedium alpha-modification (αMEM, JRH Biosciences, Lenexa, Kans.)supplemented with 5% fetal bovine serum (FBS, Gemini Bio-Products, Inc.,Calabasas, Calif.). A human epidermoid carcinoma cell line A431 (ATCCCRL1555), a human epitheloid carcinoma cell line HeLa (ATCC CRL2) and ahuman glioblastoma cell line U87MG (ATCC HTB14) were grown as monolayersin Dulbecco's modified Eagle's medium (DMEM; GIBCO-BRL, Gaithersburg,Md.) supplemented with 10% FBS. HeLa CD4⁺ Clone 1022 (NIH AIDS Researchand Reference Reagent Program), which express CD4 on their surface and ahuman Burkitt's lymphoma cell line Daudi (ATCC CCL213), (ATCC CRL1582)was maintained in RPMI 1640 (JRH Bioscience) supplemented with 10% FBS.HL-60, promyelocytic leukemia cell line (ATCC CCL240), was maintained inRPMI 1640 supplemented with 20% FBS.

[0064] Monoclonal antibodies (mAbs).

[0065] A murine mAb of IgG2a type against the human epidermal growthfactor receptor (EGFR) was obtained from Upstate Biotechnology (LakePlacid, N.Y.). Anti-HLA-DR (mouse IgG2a), anti-CD4 (mouse IgG1) andanti-CD33 (mouse IgG1) were purchased from Becton Dickinson (San Jose,Calif.).

[0066] Plasmids.

[0067] A helper plasmid DH-BB (Invitrogen Co., FIG. 1B) (Bredenbeek, P.J. et al., 1993) which contains the genes for the structural proteins(capsid, E3, E2, 6K and E1) required for packaging of the Sindbis viralgenome was used for construction of the recombinant envelope gene. ASindbis virus-based expression vector SinRep/LacZ (Invitrogen Co., FIG.3B) (Bredenbeek, P. J. et al., 1993) contains the packaging signal,nonstructural protein genes 1-4 (nsP1-4) for replicating the RNAtranscript and the lacZ gene. Plasmid pEZZ 18, which contains twosynthetic Z domains based on the B domain of protein A (Löwenadler, B.et al., 1987), was purchased from Pharmacia Biotech, Uppsala, Sweden.The phagemid pALTER-1 vector (Promega Co. Madison, Wis.) was used tointroduce the BstEII site in E2 region of DH-BB plasmid byoligo-directed site-specific mutagenesis.

[0068] Construction of the recombinant Sindbis virus structural gene.

[0069] Altered Sites in vitro Mutagenesis System (Promega Co.) was usedto introduce a specific restriction site into the E2 region of Sindbisvirus structural gene. First, a BssHII site was introduced between XbaIand HindIII sites of the pALTER-1 vector by using two compatibleoligonucleotides 5′-CTAGAGCGCGCAAA-3′ and 5′-AGCTTTTGCGCGCT-3′ (SEQ IDNOS. 6-7). A fragment between SacI and BssHII of the DH-BB plasmidcontaining the E2 region of structural gene was cloned into the pALTER-1vector. A single-stranded template of the recombinant pALTER-1 vectorwas prepared by infection of helper phage M13KO7. A mutagenicoligonucleotide (5′-ATGTCGCTTAAGCAGGTAACCACCGTTAAAGAAGGC-3′) (SEQ ID NO.8) which introduces a BstEII cloning site between codons 71 and 74 aminoacids in E2 polypeptides and an ampicillin repair oligonucleotide(5′-GTTGCCATTGCTGCAGGCATCGTGGTG-3′) (SEQ ID NO. 9) were annealed to thesingle-stranded template, followed by synthesis of the mutant strandwith T4 DNA polymerase. After transformation into E. coli, mutants wereselected in the presence of ampicillin and screened by direct sequencingof the plasmid DNA. The SacI-BssHII region of original DH-BB plasmid wasreplaced with the mutated fragment and the DH-BB-Bst plasmid wasobtained (FIG. 3B). A region of protein A (ZZ) containing two syntheticIgG-binding domain was amplified by the polymerase chain reaction (PCR)using pEZZ 18 as a template. Primers used for PCR amplification are ZZ-5(5′-CACGATGAGGTAACCGACAACAAATTCAAC-3′) and ZZ-3(5′-GGTCGAGGTTACCGGATCCCCGGGTACCGA-3′) (SEQ ID NOS. 10-11) both encodingunique BstEII sites. The resulting PCR products were digested withBstEII and inserted into predigested DH-BB-Bst plasmid at the BstEIIsite. Clones containing inserts of proper size and orientation weresequenced to confirm that the correct reading frames were maintained andthe DH-BB-ZZ plasmid was obtained (FIG. 3B). The plasmid p-DH-BB-ZZ wasdeposited with the American Type Culture Collection (ATCC) on Mar. 28,1997.

[0070] In vitro transcription and transfection for recombinant virusproduction.

[0071] Plasmids for in vitro transcription were prepared by use ofQiagen (Chatsworth, Calif.) columns. All helper plasmids (DH-BB,DH-BB-Bst and DH-BB-ZZ) and SinRep/LacZ plasmid were linearized by XhoIrestriction enzyme digestion and purified by phenol/chloroformextraction followed by ethanol precipitation. Transcription reactionswere carried out by using InvitroScript Cap Kit (Invitrogen Co.) toproduce large quantities of capped mRNA transcript from the SP6promoter. For cotransfections of helper and SinRep/LacZ RNA into BHKcells, electroporations were performed as described before (Liljeström,P. et al., 1991, Biotechnology 9:1356-1361). Electroporated cells weretransferred to 10 ml of αMEM containing 5% FCS and incubated for 12hours. Cells were then washed with PBS and incubated in 10 ml ofOpti-MEM I medium (GIBCO-BRL) without FCS. After 24 hours, culturesupernatants were harvested and aliquots were stored at −80° C.

[0072] Immunoblot assay. Cells were lysed in 20 mM Tris-HCl buffer (pH8.0) containing 1% Triton X, 0.15 M NaCl, 1 mM phenylmethylsulfonylfluoride, 1 mM EDTA and 10% glycerol 24 hour after transfection. Cellextracts were then sonicated and mixed with electrophoresis loadingbuffer (125 mM Tris-HCl, pH 6.8, 10 mM β-mercaptoethanol, 2% SDS, 10%glycerol and 0.01% bromphenol blue). Virus samples were pelleted byultracentrifugation of the supernatants (10 ml) in an SW41 BeckmannRotor (35,000 rpm, 2 h, 4° C.) and resuspended in electrophoresisloading buffer. Cell extracts and viral samples were subjected toimmunoblot analysis as described before (Marin, M. et al., 1996) byusing anti-Sindbis virus mouse immune ascitic fluid (ATCC VR-1248) andhorseradish peroxidase (HRP)-conjugated rabbit anti-goat IgG antibodies(Pierce, Rockford, Ill.).

[0073] Infection assays.

[0074] Infectivity of recombinant chimeric viruses to BHK and human celllines was determined by transfer of the Sindbis virus vector(SinRep/LacZ) that can transduce the bacterial β-galactosidase gene(Bredenbeek, P. J. et al., 1993). Viral supernatant dilutions wereincubated with or without monoclonal antibodies at room temperature for1 hour, then added to adherent (2×10⁵) and suspension (1×10⁶) cells in6-well plates. After 1 hour incubation at room temperature, cells werewashed with PBS and incubated in growth medium for 24 hours. Viralinfection was evaluated by X-Gal Staining and FACS-Gal as describedbelow and titers were estimated in LacZ CFU per milliliter.

[0075] X-Gal Staining and FACS-Gal Assay.

[0076] For X-gal staining, commercial protocol was followed. Briefly,cells were fixed in PBS containing 0.5% glutaraldehyde for 15 minfollowed by washing with PBS three times. Then cells were stained withPBS containing 1 mg/ml X-gal, 5 mM potassium ferricyanide, 5 mMpotassium ferrocyanide and 1 mM MgSO₄ at 37° C. for 2 hours. TheFACS-Gal assays were performed as described previously (Fiering, S. N.et al., 1991, Cytometry 12:291-301).

[0077] The present invention is not to be limited in scope by thespecific embodiments described which were intended as singleillustrations of individual aspects of the invention, and functionallyequivalent methods and components were within the scope of theinvention. Indeed, various modifications of the invention, in additionto those shown and described herein will become apparent to thoseskilled in the art from the foregoing description and accompanyingdrawings. Such modifications are intended to fall within the scope ofthe appended claims.

DEPOSIT OF MICROORGANISMS

[0078] The following organisms were deposited with the American TypeCulture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852 onMar. 28, 1997. Strain Designation Containing Accession No. p-439-ZZExpression plasmid p-DH-BB-ZZ Expression plasmid

[0079]

1 11 30 base pairs nucleic acid single linear DNA 1 CACGATGAGGTAACCGACAA CAAATTCAAC 30 17 base pairs nucleic acid single linear DNA 2GTTTTCCCAG TCACGAC 17 38 base pairs nucleic acid single linear DNA 3AATTCGGGAG GCGGTGGATC AGGTGGAGGC GGTTCAGG 38 36 base pairs nucleic acidsingle linear DNA 4 GATCCCTGAA CCGCCTCCAC CTGATCCACC GCCTCC 36 11 aminoacids amino acid unknown unknown protein 5 Ser Gly Gly Gly Gly Ser GlyGly Gly Gly Ser 1 5 10 14 base pairs nucleic acid single linear DNA 6CTAGAGCGCG CAAA 14 14 base pairs nucleic acid single linear DNA 7AGCTTTTGCG CGCT 14 36 base pairs nucleic acid single linear DNA 8ATGTCGCTTA AGCAGGTAAC CACCGTTAAA GAAGGC 36 27 base pairs nucleic acidsingle linear DNA 9 GTTGCCATTG CTGCAGGCAT CGTGGTG 27 30 base pairsnucleic acid single linear DNA 10 CACGATGAGG TAACCGACAA CAAATTCAAC 30 30base pairs nucleic acid single linear DNA 11 GGTCGAGGTT ACCGGATCCCCGGGTACCGA 30

What is claimed is:
 1. A virus envelope protein modified by insertion ofan IgG binding domain of Protein A.
 2. The virus envelope protein ofclaim 1, wherein the virus is an Alphavirus.
 3. The virus envelopeprotein of claim 2, wherein the Alphavirus is a Sindbis virus and theenvelope protein is an E2 protein.
 4. The virus envelope protein ofclaim 1, wherein the Fc binding domain of protein A is a ZZ domain. 5.The Sindbis virus E2 protein of claim 3, wherein the Fc binding domainof protein A is a ZZ domain.
 6. The Sindbis virus E2 protein of claim 3,wherein the Fc binding domain of protein A is inserted between aminoacid residues 71 and 74 of said E2 protein.
 7. A complex for transducinga target cell comprising a viral vector comprising a chimeric envelopeprotein containing an IgG binding domain of Protein A sufficient to bindan Fc domain of an antibody, wherein said envelope protein is a SindbisE2 protein and wherein said chimeric envelope protein alters the bindingof said E2 protein to its natural receptor; and an antibody directedagainst a surface protein on said target cell.
 8. The complex of claim7, wherein the IgG binding domain of protein A is a ZZ domain.
 9. Thecomplex of claim 7 wherein the IgG binding domain of protein A isinserted between amino acid residues 71 and 74 of said E2 protein.
 10. Aviral vector comprising an Alphavirus envelope protein modified byinsertion of an IgG binding domain of protein A.
 11. The viral vector ofclaim 10 wherein the Alphavirus is a Sindbis virus and the envelopeprotein is an E2 protein.
 12. The viral vector of claim 10 wherein theIgG binding domain of protein A is a ZZ domain.
 13. The Sindbis virus E2protein of claim 11 wherein the IgG binding domain of protein A is a ZZdomain.
 14. The E2 protein of claim 11 wherein the IgG binding domain ofprotein A is inserted between the amino acid residues 71 and 74 of saidE2 protein.
 15. An isolated nucleic acid encoding a virus envelopeprotein modified by insertion of an IgG binding domain of protein A. 16.The nucleic acid of claim 15 wherein the virus is an Alphavirus and theenvelope protein is an E2 protein.
 17. The nucleic acid of claim 15wherein the IgG binding domain of protein A is a ZZ domain.
 18. Thenucleic acid of claim 17 wherein the IgG binding domain of protein A isinserted between amino acid residues 71 and 74 of said E2 protein.